DEVELOPMENT OF A SIMPLE SLOW COOLING DEVICE FOR CRYOPRESERVATION OF SMALL BIOLOGICAL SAMPLES.

JUAN DE PAZ L.; M. CELESTE ROBERT; GRAF D.; GUIBERT E.E.; RODRIGUEZ J.V.

Rosario

Congreso; 8vo Congreso Latinoamericano de Organos Artificiales, Biomateriales e Ingeniería de Tejidos; 2014

Centro binacional (Argentina-Italia) de Investigaciones en Criobiología Clinica y Aplicada

DEVELOPMENT OF A SIMPLE SLOW COOLING DEVICE FOR CRYOPRESERVATION OF SMALL BIOLOGICAL SAMPLES. Juan de Paz L.1, Robert M.C.1, Graf D.A..2, Guibert E.E.1 and Rodriguez J.V.1* 1 Centro Binacional (Argentina-Italia) de Investigaciones en Criobiología Clínica y Aplicada (CAIC)- Universidad Nacional de Rosario. Avda. Arijón 28 bis (2000) Rosario, Argentina.2 Servicio de Electrónica y Óptica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. During the process of low temperature preservation of biological samples there is often a need for carefully controlled cooling conditions to establish the appropriate protocols to assure viability and functionality after cryopreservation. Consequently several commercial companies have developed systems for controlled freezing of cells and tissues, most of which are expensive when dealing with small number of low volume biological samples. For the present study based on the Pye´s idea (1), we designed and characterized a simple controlled-rate freezing device for the cryopreservation of a reduced number of small biological samples (up to 6 samples of 2 mL). A specific application involving the cryopreservation of neural cells is illustrated and to demonstrate that the device could be used for: to incubate the sample plus the cryoprotector agent at 10 C to allow their diffusion into cells; cooling the samples at different rates up to a final temperature of -60 C; to record the freezing curve and to determine the sample nucleation temperature (Tc) in order to monitor the entire process. The slow cooling device consist in: 1- A 2 L Dewar flask containing the refrigerant, 2- A cooling chamber containing the heat exchange medium (HEM) and a carrier to support the samples, 3- a constant rate magnetic stirrer, 4- A cylindrical cooper bar, in contact with the HEM and the refrigerant, used as heat conductor, 5- A TES-1384 electronic thermometer (TES Electrical, Taiwan) connected to a PC. In our experiments, Liquid Nitrogen (LN2) was used as refrigerant and Methanol (400 mL) was used as HEM, The heat transfer, and ultimately, the cooling rate of the bath, was fixed by selecting a proper copper bar diameter (1/4, 3/8, 1/2 and 5/8 inches diameter). To assure maximal thermal contact between the HEM and the specimen, 1.0 mL cryovials containing the thermocouple and suspensions of neuronal cells were positioned on a custom-built carrier, submerged into the methanol and cooled under constant stirring. Sample and methanol bath temperatures were measured every 10 seconds, the data collected, and a temperature vs. time graph was generated. Sample cooling rate was estimated by measuring the temperature of the tube containing a type K thermocouple and calculated by linear regression before the spontaneous crystallization peak. Also we studied the effect of HEM stirring rate (480, 780 and 960 rpm) on the cooling rate produced by different copper bars. To set up the number of samples to be cooled, we measured the HEM temperature changes produced by the release of sample latent heat of fusion. Six constant cooling rates from 0.4  0.02; 1.2  0.2; 1.9  0.1; 3.1  0.5; 4.1  0.3 and 7.0  1.9 C/min were obtained by running the system using different single and combined copper bars. A volume of 2 L of LN2 was required for each run. Tested HEM stirring rates showed no effect on cooling rate and an intermediate rate (780 r.p.m.) was selected. The appropriate number of samples positioned on the carrier was six. The proposed design is simple, and allowed the optimization of a slow cooling methodology for cryopreservation of rat neuronal cells evaluating parameters such as cooling rate and liquid nitrogen plunge temperature in relation with post-rewarming viability. The Optimal conditions for viability and function at rewarming step were achieved with cells cooled at 3.1 ± 0.5 ºC/min until sample temperature reaches ~ -50ºC and then plunged in LN2 for storage. 1- Pye, J., et al. in G.H. Zeilmaker (Ed.) Frozen Storage of Laboratory Animals. 1980 p. 61-66, Gustav Fischer Verlag, Stuttgart, New York.